PROJECT SUMMARY Understanding how information is processed in the mammalian neocortex has been a longstanding question in neuroscience. While the action potential is the fundamental bit of information, how these spikes encode representations and drive behavior remains unclear. In order to adequately address this problem, it has become apparent that experiments are needed in which activity from large numbers of neurons can be measured in a detailed and comprehensive manner across multiple timescales. Direct measurements of action potentials have primarily been achieved by electrophysiology. However, such measurements cannot easily be combined with other methods to assess the connectivity and molecular properties of neurons. Integrating functional, anatomical, and genetic information is critical for understanding how neuronal circuits are organized and computed. There have been long-standing efforts in developing optical methods for measuring neuronal activity due to its compatibility to simultaneously measure connectivity and molecular identity using fluorescent labeling techniques. Newly engineered genetically-encoded voltage-sensitive indicators have now opened the door for optical imaging of action potentials. Two-photon microscopy has been a proven method for deep non-invasive imaging into the brain. However, the fast millisecond transience of action potentials and the membrane localization of genetically-encoded voltage-sensitive indicators both contribute to conditions of limited photon flux. This creates fundamental challenges in the application of two- photon microscopy for voltage imaging that requires scanning at kilohertz frame rates with high signal to noise. To achieve this requires a concerted effort between optical engineers and protein engineers to develop new instrumentation and sensors to arrive at an optimal solution. This multi-investigator effort proposes to advance two-photon microscopy and genetically-encoded voltage-sensitive indicators to enable non-invasive population-level measurements of action potentials with single-cell spatial resolution and single-spike temporal precision deep into the mammalian brain of awake behaving animals.